The long-term objective of the proposed research is to determine the role of cultivable and uncultivable oral spirochetes in the etiology of periodontal diseases. There is substantial evidence implicating spirochetes as etiologic agents in periodontal diseases, however clinical research in this area has been stymied by difficulties in identifying and quantitating these organisms. In acute necrotizing ulcerative gingivitis (ANUG) and some forms of periodontal disease, spirochetes may comprise over 50% of the bacterial flora. Currently, there are four named oral spirochete species: Treponema denticola, T pectinovorurn, T socranskii, and T vincentii. In the first two years of this grant, we demonstrated that there are over 20 species in the periodontal pocket, but that most of these species are uncultivable by standard means. DNA probes based upon l6S rRNA sequence information have proven valuable for the direct identification and quantitation of organisms in plaque samples. We have developed DNA probes to the 4 cultivable oral treponemes and to 8 taxa of uncultivable oral spirochetes. In this proposal, we will continue to produce l6S rRNA-based DNA probes to newly identified species of cultivable and uncultivable spirochetes. In Aim l, about 250 strains provided to us by Dr. Smibert and other collaborating scientists will be screened by partial l6S rRNA sequencing. Those isolates whose sequence differs by more than 1% from sequences in our database will be fully sequenced. The l6S rRNA sequence information will be analyzed to determine species, genetic diversity, and to design DNA probes. In Aim 2, the l6S rRNA genes of oral spirochetes will be amplified directly from spirochete-rich plaque samples using polymerase chain reaction with universal and spirochete specific primers. Plaque samples will be obtained from subjects with gingivitis, ANUG and adult periodontitis, and with gingivitis and periodontitis in AIDS subjects. In Aim 3, we will examine spirochetes that grow on initial isolation, but cannot currently be maintained upon repeated passage. This aim will allow linking sequences of uncultivable spirochetes determined in Aim 2 to specific morphotypes determined by light and electron microscopy. In Aim 4, clinical studies using DNA probes in Checkerboard Hybridization will be performed to determine the prevalence of each spirochete species in subjects with gingival health, gingivitis, ANUG, and adult periodontitis. The DNA probes developed in this project will be valuable in future clinical studies to determine the role of spirochete species in each of the many types of periodontal diseases. These DNA probes will facilitate studies of spirochetal adherence, of invasion of epithelial cells, of oral ecology, and of spirochetal pathogenic mechanisms. This project will also facilitate studies in the medical community of spirochetes elsewhere in the body, and of spirochete superinfections in subjects with AIDS.